Báo cáo khoa học: Investigation of the effects of sulfonylurea exposure on pancreatic beta cell metabolism pptx

Investigation of the effects of sulfonylurea exposure on pancreatic beta cell metabolism Lorraine Brennan 1 , Chandralal Hewage 1 , J. P. G. Malthouse 1 , Neville H. McClenaghan 2 , Peter R. Flatt 2 and Philip Newsholme 1 1 UCD School of Biomolecular and Biomedical Science, UCD Conway Institute, University College Dublin, Belfield, Ireland 2 School of Biomedical Sciences, University of Ulster, Coleraine, UK Sulfonylureas are a major class of potent insulinotrop- ic drugs that are used extensively in the treatment of patients with type 2 diabetes. These drugs stimulate secretion of insulin from the pancreatic beta cell, primarily by interacting with the high-affinity sulfonyl- urea receptor (SUR1) subunit of the beta cell ATP-sensitive potassium ion channel (K ATP ) channel. Sulfonylurea drugs differ in their specificity for the SUR1 subunit. The interaction with the K ATP channel closes the channel, causing membrane depolarization and subsequent opening of voltage-dependent Ca 2+ channels [1]. The resulting influx of Ca 2+ leads to a rapid rise in intracellular Ca 2+ concentration and trig- gers insulin secretion [2,3]. However, a growing body of evidence suggests that the sulfonylureas additionally act in a K ATP channel-independent manner by directly interacting with the secretory machinery, and it has been suggested that this effect is indirectly dependent on protein kinase C activation [4–6]. Interestingly, a recent study has proposed a mechanism by which sulfonylureas could be transported across the plasma membrane and ultimately interact with intracellular sites regulating insulin exocytosis [7]. Treatment of patients with sulfonylureas for pro- longed periods (several years) often results in impaired sulfonylurea-induced insulin secretion [8,9]. Although the exact reasons underlying this phenomenon remain unclear, it is now believed that this may be at least partly attributed to desensitization of the pancreatic beta cells to the actions of these drugs. Experiments investigating the effects of chronic exposure of pancre- atic islets and beta cell lines to sulfonylureas in vitro have shown that the desensitization in certain cases is not limited to subsequent drug-induced insulin Keywords beta cells; metabolism; sulfonylurea Correspondence L. Brennan, UCD School of Biomolecular and Biomedical Science, UCD Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland Fax: +353 1 2837211 Tel: +353 1 7166781 E-mail: lorraine.brennan@ucd.ie (Received 9 June 2006, revised 21 Septem- ber 2006, accepted 22 September 2006) doi:10.1111/j.1742-4658.2006.05513.x Prolonged exposure of pancreatic beta cells to the sulfonylureas glibenca- mide and tolbutamide induces subsequent desensitization to the actions of these drugs. The precise mechanisms underlying this desensitization remain unknown, prompting the present study, which investigated the impact of prolonged sulfonylurea exposure on glucose and energy metabolism using clonal pancreatic BRIN-BD11 beta cells. Following prolonged exposure to tolbutamide, BRIN-BD11 beta cells were incubated in the presence of [U- 13 C]glucose, and isotopomer analysis revealed that there was a change in the ratio of flux through pyruvate carboxylase (EC 6.4.1.1) and pyruvate dehydrogenase (EC 1.2.4.1, EC 2.3.1.12, EC 1.8.1.4). Energy status in intact BRIN-BD11 cells was determined using 31 P-NMR spectroscopy. Exposure to tolbutamide did not alter the nucleotide triphosphate levels. Collectively, data from the present study demonstrate that prolonged expo- sure of beta cells to tolbutamide results in changes in flux through key enzymes involved in glucose metabolism that, in turn, may impact on glucose-induced insulin secretion. Abbreviations K ATP channel, ATP-sensitive potassium ion channel; NTP, nucleotide triphosphate; PC, pyruvate carboxylase; PDH, pyruvate dehydrogenase. 5160 FEBS Journal 273 (2006) 5160–5168 ª 2006 The Authors Journal compilation ª 2006 FEBS secretion, but may also affect nutrient-induced insulin secretion [10–13]. The precise mechanisms underlying beta cell drug-induced desensitization remains elusive, and it remains unclear as to whether it results from depletion of insulin stores or functional changes in signalling pathways [14]. In a study investigating desensitization due to chronic exposure to glibenca- mide in MIN6 cells, a decrease in the number of fun- ctional K ATP channels on the plasma membrane was observed [15]. This would support a homologous desensitization theory, where the primary cause of the desensitization is due to the occupancy of receptor sites and subsequent modification of the downstream pathways [14]. However, other studies have demon- strated that prolonged treatment with sulfonylurea drugs affects the subsequent secretory response to other stimuli [12,13,16], indicating the presence of heterogeneous desensitization. Acute stimulation of insulin secretion by glucose involves metabolism via oxidative and anaplerotic pathways involving pyruvate dehydrogenase (PDH; EC 1.2.4.1, EC 2.3.1.12, EC 1.8.1.4) and pyruvate carboxylase (PC; EC 6.4.1.1) [17–19]. The resultant increase in ATP concentration closes the K ATP chan- nels, resulting in membrane depolarization, opening of the voltage-dependent Ca 2+ channels, Ca 2+ influx, and a subsequent increase in cytosolic Ca 2+ ([Ca 2+ ] c ) which in turn triggers insulin secretion. Additionally, the influx of Ca 2+ results in an uptake of Ca 2+ by the mitochondria, which may activate mitochondrial meta- bolism and result in further increases in ATP concen- tration [20–22]. The present study investigated the effects of prolonged exposure to sulfonylurea drugs on beta cell metabolism using a combination of different NMR techniques, and utilizing the well-characterized clonal glucose-responsive pancreatic beta cell line, BRIN-BD11 [23,24]. Results Metabolism of [1- 13 C]glucose in the presence and absence of sulfonylurea(s) Following 18 h of prior culture in the presence of tol- butamide or glibencamide, the cells were incubated for 1 h in the presence of [1- 13 C]glucose with or without the sulfonylurea drug. The main metabolites produced included glutamate labelled at positions C2, C3 and C4, lactate and alanine labelled at position C3, and aspartate labelled at positions C2 and C3. Comparison of the label distribution between the different treat- ments indicated that there was no significant change in the total concentration of glutamate labelled at position C4 (Table 1). In the case of tolbutamide pre- treatment, there was a general trend for decreased labelling of glutamate in positions C2 and C3, but the changes were significant only for the C2 position. The total 13 C concentration of alanine and lactate labelled at position C3 did not change significantly. However, in the presence of tolbutamide, the total amount of 13 C label present in aspartate C2 and C3 increased significantly. The percentage enrichments of the glutamate peaks in the presence and absence of the drugs are reported in Table 2. No significant changes were observed for glutamate C3 and C4. In the presence of tolbutamide, there was a decrease in the percentage enrichment for glutamate C2. The amount of glucose remaining in the medium was measured in the presence and absence of drugs, and was found to be significantly different only in the presence of glibencamide; under control conditions, the amount of glucose remaining was 19.5 ± 1.6 lmolÆmg )1 protein, whereas in the presence of glibencamide, the concentration was 14.3 ± 0.9 lmolÆmg )1 protein (P<0.005). The concentration of glucose remaining in the medium after tolbutamide exposure was 17.9 ± 2.1 lmolÆmg )1 protein. The insulin released into the medium at the end of a 1 h incubation period following the preculture with and without the drugs Table 1. Amount of 13 C-labelled amino acids present after incuba- tion for 1 h with [1- 13 C]glucose in the presence and absence of sul- fonylurea drugs. Values are given as nmol per mg protein ± SD (n ¼ 3). All experiments were carried out after a preincubation per- iod of 18 h in the presence or absence of drug. Control + Tolbutamide + Glibencamide Glu C2 5.8 ± 1.0 3.6 ± 0.4* 5.7 ± 0.6 Glu C3 5.1 ± 1.0 3.8 ± 0.8 6.0 ± 1.1 Glu C4 15.8 ± 2.0 12.5 ± 1.0 17.9 ± 3.2 Lactate C3 7.5 ± 2.6 6.1 ± 2.8 6.2 ± 1.4 Alanine C3 4.8 ± 1.3 4.6 ± 1.3 4.1 ± 0.6 Asp C2 2.4 ± 0.5 4.0 ± 0.4* 2.6 ± 1.3 Asp C3 2.8 ± 0.2 4.9 ± 0.8* 2.6 ± 1.3 *P<0.05. Table 2. Percentage enrichment in the glutamate carbons following incubation for 1 h with [1- 13 C]glucose in the presence and absence of sulfonylurea drugs. Values are expressed as a percentage ± SD (n ¼ 3). All experiments were carried out after a preincubation period of 18 h in the presence or absence of drug. Control + Tolbutamide + Glibencamide Glu C2 8.4 ± 0.6 6.6 ± 0.5* 8.3 ± 0.6 Glu C3 7.4 ± 1.6 7.0 ± 2.3 8.7 ± 1.2 Glu C4 22.8 ± 1.3 23.0 ± 3.0 26.0 ± 1.6 *P < 0.05. L. Brennan et al. Metabolic effects of sulfonylurea exposure FEBS Journal 273 (2006) 5160–5168 ª 2006 The Authors Journal compilation ª 2006 FEBS 5161 was measured. Preincubation in the presence of the drugs significantly reduced the amount of insulin released (Fig. 1). Effects of tolbutamide and glibencamide on [U- 13 C]glucose metabolism To probe further the effect of tolbutamide and to investigate the observed increase in aspartate produc- tion, flux analysis was carried out using [U- 13 C]glu- cose. Following prior culture for 18 h in the presence of tolbutamide, BRIN-BD11 cells were incubated in the presence of tolbutamide and [U- 13 C]glucose for 2 h. Control experiments were run in parallel with no drug present. A typical spectrum obtained from con- trol cellular extracts is shown in Fig. 2. There was no significant change in the total amount of labelled glu- tamate, lactate or alanine produced following culture with tolbutamide (data not shown). The ratio of the flux through PC and PDH was determined in the pres- ence and absence of the drug, as described in Experi- mental procedures (Table 3). A significant reduction in the ratio was found in the presence of tolbutamide. The fraction of acetyl-CoA derived from [U- 13 C]glu- cose was determined, and is reported in Table 4. Preincubation with tolbutamide did not change the percentage significantly, indicating that the fraction of glucose entering the tricarboxylic acid cycle via the PDH-mediated conversion to acetyl-CoA did not 0 1 2 3 4 5 6 7 control tol glib Insulin release [pmol·(mg protein) –1 ] * * Fig. 1. Effects of culture (18 h) with 100 lM tolbutamide (Tol) or 1 l M glibencamide (Glib) on glucose-induced insulin release over a subsequent 1 h incubation period. Values are mean ± SD. Control conditions refer to culture and subsequent incubation in the absence of a drug. *P < 0.05. Dioxane Glucose Glu C2 Glu C4 Glu C3 Lac C3 Ala C3 Ala C2 Acetate C2 Glucose 80 70 60 50 40 30 20 Chemical Shift (p.p.m.) Fig. 2. A typical 13 C-NMR spectrum obtained for control conditions after incubation in the presence of 8.4 mM [U- 13 C]glucose for 2 h. Lac, lactate. Table 3. Ratio of flux through pyruvate carboxylase (PC) and pyru- vate dehydrogenase (PDH) calculated from C2 and C4 resonances of glutamate in the presence and absence of a sulfonylurea drug. For all experiments, cells were incubated in the presence of [U- 13 C]glucose for 2 h following 18 h of prior culture in the presence or absence of the drug. Values are expressed as averages ± SD. Condition PC ⁄ PDH Control (n ¼ 3) 0.34 ± 0.04 Tolbutamide (n ¼ 3) 0.24 ± 0.02* Glibencamide (n ¼ 2) 0.30 ± 0.01 *P < 0.05. Table 4. Percentage of acetyl-CoA derived from [U- 13 C]glucose fol- lowing incubation in the presence and absence of a sulfonylurea drug. Values are expressed as a percentage ± SD. Cells were incu- bated in the presence of [U- 13 C]glucose with or without a drug for 2 h following a preincubation period of 18 h in the presence or absence of a drug. Condition Percentage labelled from [U- 13 C]glucose Control (n ¼ 3) 66 ± 10 Tolbutamide (n ¼ 3) 65 ± 5 Glibencamide (n ¼ 2) 76 ± 1 Metabolic effects of sulfonylurea exposure L. Brennan et al. 5162 FEBS Journal 273 (2006) 5160–5168 ª 2006 The Authors Journal compilation ª 2006 FEBS change. Thus, the observed change in the ratio of flux through PC and PDH must have been due to changes in anaplerotic metabolism through PC. When cells were preincubated in the presence of glibencamide followed by [U- 13 C]glucose, the ratio of the flux through PC and PDH did not differ signifi- cantly from the control value. 31 P-NMR studies of intact pancreatic BRIN-BD11 beta cells A typical spectrum obtained from cells grown on fibra- cel beads in a mini-bioreactor is shown in Fig. 3. The T1 (longitudinal relaxation) values for the NTP peaks were calculated using inversion recovery experiments. The following values were found: a, 0.65 ± 0.14 s; b, 0.55 ± 0.05 s; and c, 0.88 ± 0.19 s. Control condi- tions represent conditions where cells were maintained in standard RPMI-1640 medium supplemented with 2mm glutamine, 10% (v ⁄ v) fetal bovine serum, and 0.1% antibiotics. When tolbutamide was added to the culture medium to give a final concentration of 100 lm, there were no significant changes in the nuc- leotide triphosphate (NTP) levels over a 24 h period (Fig. 4). The free cytoplasmic ADP concentration is directly proportional to the phosphocreatine ⁄ ATP ratio [25,26]. There was no significant change in this ratio on addition of tolbutamide, which, in the absence of a change in the intracellular pH or total creatine pool, indicated that there was no change in free ADP concentration. Discussion Previous studies using clonal pancreatic BRIN-BD11 beta cells have demonstrated that prolonged exposure (18 h) to sulfonylureas induces specific and readily reversible desensitization to subsequent treatment with the drugs [13,27]. It has also been reported that chro- nic exposure (72–144 h) can result in an irreversible concentration- and time-dependent decline in sulfonyl- urea-induced insulin secretion [28]. As the mechanisms underlying the latter effects remain to be determined, the principal aim of this study was to investigate the impact of sulfonylurea exposure on cellular metabo- lism using clonal pancreatic BRIN-BD11 beta cells. Under the experimental conditions used in this study, there was a significant decrease in acute insulin release following preincubation with the drugs compared to control conditions. Previous studies on tolbutamide have demonstrated that under the same conditions as described in this study, the insulin content does not change [27]. Prolonged exposure to tolbutamide and glibenca- mide did not significantly alter the amount of glu- tamate labelled at positions C4 and C3 following a 1 h incubation with [1- 13 C]glucose. The amount of Chemical Shift (p.p.m.) 20 10 0 -10 -20 MDP PCR Pi PME NTP Fig. 3. A typical 31 P-NMR spectrum of intact BRIN-BD11 cells grown in the mini-bioreactor. MDP, methylene diphosphonate; PME, phospho- monoesters; Pi, inorganic phosphate; PCR, phosphocreatine; NTP, nucleotide triphosphate. 0 0.1 0.2 0.3 0.4 0.5 0.6 0 5 10 15 20 25 30 35 40 β NTP/MDP control tolbutamide time (h) Fig. 4. Nucleotide triphosphate levels in intact BRIN-BD11 cells from a representative culture of cells. After a control period of 15 h, the cells were perfused in medium with 100 l M tolbutamide for 24 h. L. Brennan et al. Metabolic effects of sulfonylurea exposure FEBS Journal 273 (2006) 5160–5168 ª 2006 The Authors Journal compilation ª 2006 FEBS 5163 label at the C2 position showed a small decrease fol- lowing incubation in the presence of tolbutamide. The amount of labelled lactate and alanine did not significantly change following incubation with either drug. However, following prolonged exposure to tol- butamide, the amount of aspartate labelled at posi- tions C2 and C3 significantly increased. Aspartate can be formed via a transamination reaction with oxaloacetate, which is itself produced from pyruvate via a reaction catalysed by PC. Oxaloacetate is in equilibrium with malate, which can leave the mitoch- ondrial matrix and in the cytosol is converted back to pyruvate via malic enzyme (pyruvate cycling). To gain a better understanding of these changes, experi- ments were subsequently performed using [U- 13 C]glu- cose, and analysis of the isotopomers formed allowed calculation of the fluxes through specific enzymes (Fig. 5). A decrease in the ratio of fluxes through PC and PDH (PC ⁄ PDH) was determined following 18 h of exposure to tolbutamide. However, there was no change in the fraction of acetyl-CoA labelled from [U- 13 C]glucose via the PDH pathway, indicating that the change in ratio was attributable to a reduction in the flux through PC. Notably, other studies investi- gating the acute effects of glibencamide and megliti- nide on glucose oxidation in mouse pancreatic islets found no inhibitory effects, consistent with our obser- vations that oxidative pathways of glucose metabolism remain unchanged [29]. In recent years, the importance of flux through anaplerotic pathways in beta cells has been highlighted. Cline et al. [30] reported a strong positive correlation between insulin secretion and PC flux in INS-1 cells. Furthermore, Lu et al. demonstra- ted that the responsiveness of INS-1 cells to glucose- stimulated insulin secretion was linked to the cycling [31] of pyruvate (i.e. the flux through the pyruvate– malate and pyruvate–citrate cycles). In a recent study, Fransson et al. showed, by use of a PC inhibitor, the importance of flux through PC for responsiveness to glucose [32]. In the present study, the observed decrease in flux through PC observed after prolonged exposure to tolbutamide may be a contributary factor to the resulting drug-induced desensitization to acute stimulation by glucose. The present data also indicate differential effects of the two sulfonylureas on beta cell metabolism, as glib- encamide did not change end-product concentrations or the ratio of fluxes through PC and PDH. Preincu- bation with either of the drugs resulted in reduction of subsequent glucose-stimulated insulin release, suggest- ing that there is not a common mechanism of desensi- tization. However, in the present studies, a relatively low concentration of glibencamide (1 l m) was used, due to the reported higher affinity of this drug for K ATP channels [33] compared to tolbutamide. Glyb- encamide at 1 lm induces a similar secretory response to that observed for 100 lm tolbutamide [16,34,35]. Early studies investigating the effects of sulfonyl- ureas on ATP levels reported reduced concentrations in islets [36–38]. More recently Elmi et al. [29] showed that glibencamide reduced ATP concentration in the absence of glucose, but the effects were not observed at 10 mm glucose. These observations were used to suggest that the reduced ATP levels may result from increased consumption of ATP by activation of the Na + ⁄ K + pump. This is consistent with the results of a 31 P-NMR study of intact b-HC9 cells, which revealed OAA citrate α-KG malate [U- 13 C]glucose pyruvate Acetyl-CoA PC PDH PDH PC glu glu glu PC aspartate 1 2 3 4 5 Fig. 5. Overview of the metabolism of [U- 13 C]glucose. For simplicity, only the iso- topomers of glutamate formed after one turn of the tricarboxylic acid cycle (TCA) are shown. The filled circles represent labelling of the carbon position with 13 C. [U- 13 C]Pyru- vate enters the TCA cycle via pyruvate dehydrogenase (PDH), forming [1,2- 13 C]acetyl-CoA and consequently [4,5- 13 C]glutamate. If pyruvate enters via pyruvate carboxylase (PC), two 13 C isotopo- mers of oxaloacetate are derived, [1,2,3- 13 C]oxaloacetate and [3,4- 13 C]oxalo- acetate, consequently leading to [2,3- 13 C]glutamate and [1,2,3- 13 C]glutamate [48]. Glu, glutamate; OAA, oxaloacetate; a-KG, a-ketoglutarate. Metabolic effects of sulfonylurea exposure L. Brennan et al. 5164 FEBS Journal 273 (2006) 5160–5168 ª 2006 The Authors Journal compilation ª 2006 FEBS that ATP levels did not change on addition of the sul- fonylurea glyburide at glucose concentrations of 5 mm [39]. However, another study using MIN-6 cells showed that if the cells were initally primed with tol- butamide (200 lm), the subsequent increase in ATP in response to 30 mm glucose was potentiated [40]. All of these studies are distinct from ours, in that they focused on the effects of acute sulfonylurea exposure, whereas we examined the effects of prolonged exposure on ATP levels using 31 P-NMR. Our data are consistent with other studies, which reported no alteration in cel- lular ATP levels after addition of sulfonylureas in the presence of glucose. Collectively, the present data demonstrate novel changes to fluxes in glucose metabolism following pro- longed exposure to the important sulfonylurea drug tolbutamide, resulting in a 25% decrease in the PC ⁄ PDH ratio. Furthermore, these data reveal a reduction in anaplerotic flux through PC. These obser- vations are notable in that they raise the possibility that chronic sulfonylurea exposure in vivo may impact on glucose metabolism, which may contribute to the reported phenomena of sulfonylurea desensitization and, indeed, sulfonylurea failure in type 2 diabetes. Future studies determining the molecular mechanisms of tolbutamide-mediated reduction in flux through PC may lead to the design of more effective insulinotropic drugs in the future. Experimental procedures Reagents d-[1- 13 C]Glucose and d-[U- 13 C]glucose were obtained from Goss Scientific (Great Baddow, Essex, UK). All other chemicals were obtained from Sigma-Aldrich Chemical Company (Poole, Dorset, UK). Culture media and fetal bovine serum were obtained from Gibco (Glasgow, UK). Treatment of BRIN-BD11 cells with drugs Pancreatic BRIN-BD11 beta cells were utilized in these studies [41], representing a particularly useful model in which to conduct extensive NMR studies [23,24]. BRIN- BD11 cells were maintained in RPMI-1640 tissue culture medium with 10% (v ⁄ v) fetal bovine serum, 0.1% antibiot- ics (100 UÆmL )1 penicillin and 0.1 mgÆmL )1 streptomycin) and 11.1 mmd-glucose (pH 7.4). The cells were maintained at 37 °C in a humidified atmosphere of 5% CO 2 and 95% air using a Forma Scientific (Waltham, MA) incubator. For experiments on prolonged exposure to sulfonylureas, the cells were grown in T175 flasks and treated for 18 h in the presence of the drug at the specified concentration (tolbutamide 100 lm and glibencamide 1 lm). Cells were then washed with NaCl ⁄ P i and preincubated at 37 °C for 20 min in Krebs ⁄ Ringer bicarbonate buffer with 1.1 mm d-glucose (115 mm NaCl, 4.7 mm KCl, 1.28 mm CaCl 2 , 1.2 mm KH 2 PO 4 , 1.2 mm MgSO 4 .7H 2 O, 10 mm NaHCO 3 , 5gÆL )1 BSA, pH 7.4). This was followed by incubation in the presence of 8.4 mm labelled glucose ([1- 13 C]glucose or [U- 13 C]glucose) and drug for a specified period (1 h or 2 h). Control experiments were carried out in parallel in the absence of the drug. Following the incubation period, the medium was removed and stored at ) 20 °C. Subsequently, the glucose concentration and the insulin released were measured. The insulin released was measured using a Mercodia (Uppsala, Sweden) ultrasensitive rat insulin ELISA. The metabolites were extracted using a perchloric acid extraction procedure. Briefly, the cells were washed with ice-cold NaCl ⁄ P i . Per- chloric acid (6%) was added, and the cells were scraped off the culture flasks. The extracts of six culture flasks (approximately 10 8 cells) were pooled and centrifuged at 200 g with a Sigma 2-15 rotor. The resulting supernatant was neutralized with KOH (5 m and 0.1 m solutions), and the pellets were soaked overnight in 0.1 m NaOH. The pro- tein concentration was determined using the Lowry method [42]. The neutralized supernatant was centrifuged (200 g with a Sigma 2-15 rotor), and the supernatant was treated with Chelex-100 resin and then lyophilized. Each experi- ment was carried out on at least two independent cultures of the BRIN-BD11 cells. The lyophilized cell extracts were dissolved in 3 mL of potassium phosphate buffer (100 mm, pH 7.0) and then centrifuged at 200 g with a Sigma 2-15 rotor. The supernatant was carefully removed, 10% D 2 O was added, and the pH was checked and adjusted when necessary with 0.1 m NaOH and 0.1 m HCl. NMR experi- ments were performed as detailed below. Culture of BRIN-BD11 cells at high density in a mini-bioreactor A system was set up to monitor energy metabolism in intact BRIN-BD11 cells as previously described [43]. Briefly, it consists of a 2 L stirred tank fermenter in which the temperature, pH, CO 2 content and dissolved O 2 content of the culture medium were monitored and maintained within a specified range. The mini-bioreactor is a purpose- built bioreactor consisting of a capped polysulfone tube (10 mm diameter) with an inlet tube and an outlet tube. Cells were maintained attached to fibracel disks (New Brunswick Scientific, Edison, NJ) in the mini-bioreactor and were perfused with medium from the fermenter, which was pumped to the mini-bioreactor in the NMR magnet via water-jacketed tubing at 37 °C. Medium in the ferment- er was replaced by fresh medium using a ‘feed and bleed’ system. By adjustment of the feed and bleed system, L. Brennan et al. Metabolic effects of sulfonylurea exposure FEBS Journal 273 (2006) 5160–5168 ª 2006 The Authors Journal compilation ª 2006 FEBS 5165 nutrients can be maintained at a certain level. For seeding of the bioreactor, the cells were pumped into the mini-bio- reactor as a slow rate over a period of 2 h. Typically 1 · 10 8 to 3 · 10 8 cells were passed through the carriers, and attachment of 85–90% of the cells was routinely found. NMR spectroscopy 13 C-NMR of perchloric acid extracts For 13 C-NMR experiments, an insert containing 5% v ⁄ v dioxane in water was used as an external signal intensity reference. A solution of l-alanine, l-glutamate, lactate and d-glucose, each at a concentration of 100 mm, was prepared and used to quantitate concentrations of metabolites in the 13 C spectra. Proton-decoupled 13 C spectra were acquired on a Bruker (Karlsruhe, Germany) DRX 500 spectrometer using a 10 mm broadband probe. Typically, spectra were acquired with 32 000 data points using 90° pulses, 260 p.p.m. spectral width, 2.5 s relaxation delay, and 12 000–20 000 scans. Spectra were recorded at 25 °C. Chemical shifts were referenced to tetramethylsilane at 0 p.p.m. Data were processed with no zero filling using Bruker winnmr software, and exponential multiplications with 2 Hz line broadening were performed. The assign- ments of the intermediate metabolites were made by com- parison with chemical shift tables in the literature [44] or by addition of 100 mm unlabelled amino acid. The amount of 13 C in each resonance was evaluated by integration of the extract peaks and the corresponding peaks in the stand- ard sample relative to the dioxane signal. Corrections for the natural abundance signal were made. In the case of the aspartate peaks, the amount of 13 C was estimated by use of the integrals and the known dioxane concentration. The contributions of the individual isotopomers were assessed using the deconvolution routines in winnmr. The absolute enrichments of the l-glutamate were related to the glutam- ate concentration in the extracts, determined by enzymatic methods, to give the specific enrichments [45]. The fluxes reported were obtained by analysis of the isotopomers of glutamate C2 and C4. The ratio between flux through PC and PDH was calculated as follows ([2,3- 13 C 2 ]+ [1,2,3- 13 C 3 ]) ⁄ [4,5- 13 C 2 ] + [3,4,5- 13 C 3 ] [46]. The fraction of acetyl-CoA labelled from [U- 13 C]glucose was calculated using the following equation for the C4 peak: (3,4,5- 13 C) · C4 ⁄ C3 [47]. 31 P-NMR of intact cells Following removal of the upper barrel of the magnet, the mini-bioreactor was carefully guided into the probe head for the collection of NMR data with the aid of a custom- built holder. Typically, spectra were acquired with 8000 data points using 90° pulses, 50 p.p.m. spectral width, 2.0 s relaxation delay, and 512 scans. Spectra were recorded at 37 °C. Chemical shifts in aqueous media were referenced to methylene diphosphonate at 17.0 p.p.m., which was contained in a sealed capillary in the mini-bioreactor. Expo- nential multiplications with 20 Hz line broadening were performed using winnmr software. The three phosphate groups in NTPs give three distinct peaks. These peaks were primarily attributed to ATP. The T1 values of the NTP peaks were determined using inversion recovery experiments in three independent sets of cells, and these values were then used to optimize the NMR acquisition parameters. To investigate the effects of tolbutamide on energy metabolism, the cells were perfused with medium containing 100 lm tol- butamide for a period of 24 h. Spectra were recorded every 40 min. In some cases, slight acidification of the bioreactor environment was apparent by appearance of a shoulder on the P i peak attributed to intracellular P i . When this occurred, the flow rate through the bioreactor was increased as previously described [43]. Statistical analysis The results are presented as mean ± SD for n separate determinations. Groups of data were compared using Student’s unpaired t-test. Differences were considered signi- ficant at P < 0.05. Acknowledgements LB was in receipt of a Health Research Board of Ireland postdoctoral fellowship, which is gratefully acknowledged (PD ⁄ 2002 ⁄ 9). References 1 Aguilar-Bryan L, Nichols CG, Wechsler SW, Clement JPT, Boyd AE 3rd, Gonzalez G, Herrera-Sosa H, Nguy K, Bryan J & Nelson DA (1995) Cloning of the beta cell high-affinity sulfonylurea receptor: a regulator of insulin secretion. 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Brennan et al. 5168 FEBS Journal 273 (2006) 5160–5168 ª 2006 The Authors Journal compilation ª 2006 FEBS . desensitization of the pancreatic beta cells to the actions of these drugs. Experiments investigating the effects of chronic exposure of pancre- atic islets and beta cell lines to sulfonylureas. 2006) doi:10.1111/j.1742-4658.2006.05513.x Prolonged exposure of pancreatic beta cells to the sulfonylureas glibenca- mide and tolbutamide induces subsequent desensitization to the actions of these drugs. The precise mechanisms. [20–22]. The present study investigated the effects of prolonged exposure to sulfonylurea drugs on beta cell metabolism using a combination of different NMR techniques, and utilizing the well-characterized clonal